Mir396b Negatively Regulates Eggplant Defense Response to Verticillium Wilt

doi:10.16420/j.issn.0513-353x.2021-0569

Acta Horticulturae Sinica ›› 2022, Vol. 49 ›› Issue (8): 1713-1722.doi: 10.16420/j.issn.0513-353x.2021-0569

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Mir396b Negatively Regulates Eggplant Defense Response to Verticillium Wilt

LIN Yuanmi, ZHU Wenjiao, CHEN Min, XUE Chunmei, JIN Fangyu, ZHU Yuping, JIANG Xinyue, YE Lingfeng, NI Shunanling, YANG Qing^*()

College of Life Science,Nanjing Agricultural University,Nanjing 210095,China

Received:2021-12-27 Revised:2022-03-07 Online:2022-08-25 Published:2022-09-05
Contact: YANG Qing E-mail:qyang19@njau.edu.cn

Abstract

Abstract:

In order to identify the function of miR396b in eggplant resistance to verticillium wilt,pri-miR396b gene was cloned from eggplant cultivar‘Suqi 1’and miR396b transgenic eggplant lines were obtained through Agrobacterium-mediated transformation. In infected eggplant,the expression level of miR396b was down-regulated by Verticillium dahliae induction. The disease resistance analysis showed that miR396b overexpression line was more sensitive to Verticillium dahliae infection compared with the control with disease index of 75.2,while miR396b antisense lines showed more resistance with disease index of 16.2-25.3. ITS quantitative analysis showed that the content of Verticillium dahliae in the vascular tissues of miR396b overexpression plants was significantly increased compared with the wild- type eggplant,while that in miR396b antisense plants was significantly reduced. Antioxidant enzyme activity analysis showed that the activities of SOD,POD and CAT in miR396b overexpression line were lower than those in the wild-type eggplant,while they were higher in antisense lines than those in the wild-type eggplant. These results showed that miR396b might play a negative role in eggplant defense response against Verticillium wilt.

Key words: eggplant, miR396b, verticillium wilt, functional analysis

CLC Number:

S641.1

LIN Yuanmi, ZHU Wenjiao, CHEN Min, XUE Chunmei, JIN Fangyu, ZHU Yuping, JIANG Xinyue, YE Lingfeng, NI Shunanling, YANG Qing. Mir396b Negatively Regulates Eggplant Defense Response to Verticillium Wilt[J]. Acta Horticulturae Sinica, 2022, 49(8): 1713-1722.

Figures/Tables 9

Table 1 Primers used in this study

名称 Name	序列（5′-3′） Sequence	名称 Name	序列（5′-3′） Sequence
pri-miR396b-F	GAAGATCTCGAAAGAATATGTCCCAAAGTG	ITS-R	CGCCTGCGGGACTCCGATGCGAGCTGTAAC
pri-miR396b-R	GGACTAGTTGGCTGTTTGATGACGATGAC	α-tublin-F	CCACCATCAAGACTAAGCG
pri-miR396b-anti-F	GGACTAGTCGAAAGAATATGTCCCAAAGTG	α-tublin-R	AGAAGACCTCAGCAACACTC
pri-miR396b-anti-R	GAAGATCTTGGCTGTTTGATGACGATGAC	qRT-SmGRF2-F	GATCTACAAGCATTTCGTGGCA
miR396b-R	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAAAGTTC	qRT-SmGRF2-R	ATCAGTGTTGCCAGCGAAAC
miR396b-F	GAGTGTTCCACAGCTTTCTT	SmEF1a-F	CCACACTTCTCATATTGCTGTCA
Uni-R	GTGCAGGGTCCGAGGT	SmEF1a-R	ACCAGCATCACCATTCTTCAAAA
U6-F	CGGGGACATCCGATAAAA	Veri-pri-396b-F	GGCTTGATTATCTTCAAGTACTTCC
U6-R	TTGGACCATTTCTCGATTTG	Veri-pri-396b-anti-F	GGAAGTACTTGAAGATAATCAAGCC
ITS-F	CCGCCGGTCCATCAGTCTCTCTGTTTATAC	Veri-1305-R	CCGTATGTTGCATCACCTTCAC

Fig. 1 Expression analysis of eggplant miR396b under the stress of Verticillium dahliae * P﹤0.05,** P﹤0.01.

Fig. 2 Cloning of pri-miR396b and vector construction A：Agarose gel electrophoresis of PCR products of pri-miR396b gene. B：Construction of miR396b overexpression vector,Ⅰ-Enzyme digestion verification,Ⅱ-PCR identification of recombinant plasmid in transformed E. coli,Ⅲ-PCR identification of recombinant plasmid in transformed Agrobacterium.

Fig. 3 Regeneration of transgenic eggplant plants A：Regeneration of miR396b overexpression plants;B：Regeneration of miR396b antisense plants.

Fig. 4 PCR identification of miR396b-overexpressing eggplants（A）and antisense eggplants（B）,and qRT-PCR analysis of miR396b（C） M：DL2000marker;WT：wild-type plants;P：positive plasmid control;1305：plants transformed with blank 1305 plasmid;OE-1：miR396b overexpression plants;AE-1,AE-2,AE-3：miR396b antisense plants. The same below.

Fig. 5 Analysis of the phenotype and disease index of transgenic eggplants after Verticillium dahliae inoculation

Fig. 6 qRT-PCR analysis of fungal colonization of Verticillium dahliae internal transcribed spacer（ITS）transcript levels in transgenic eggplant of miR396b

Fig. 7 Analysis on the activities of antioxidant enzymes in miR396b transgenic eggplant

Fig. 8 Expression analysis of miR396b target SmGRF2 under Verticillium dahliae stress

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Mir396b Negatively Regulates Eggplant Defense Response to Verticillium Wilt

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